As compared with conventional analytical methods, the radioimmunoassay offers several advantages, particularly extreme sensitivity and high specificity. With conventional analytical methods, it is difficult to measure the minute amounts of digoxin in serum, usually less than five nanograms per milliliter (ng/ml). Radioimmunoassay procedures are based upon the characteristics that an antibody binds equally to labeled or unlabeled antigen, for example. The concentration of the nonlabeled form in the solution determines the relative amount of labeled or nonlabeled antigen which will bind to antibody. By keeping the concentration of antibody and labeled antigen constant and conducting the radioimmunoassay procedure using a series of known amounts of nonlabeled antigen, a standard curve can be constructed. Subsequently, when an unknown amount of antigen in a serum sample is reacted in the same way, its concentration can be determined by relating the value obtained to the standard curve. Accordingly, levels of antigen in serum may be measured in nanograms.
There are four requisites for a radioimmunoassay procedure: (1) a highly purified antigen; (2) a specific antibody (antiserum) produced by an injection of that antigen into another animal species; (3) radioactively labeled purified antigen or antigen derivative having a previously determined specific activity; and (4) a satisfactory method for the separation of the antigen-antibody complex from the free antigen. Free antigen can be separated from the antigen bound to the antibody and, depending on the method of separation, either free or bound antigen can be measured by determining the radioactivity.
Yalow, R.S. and Berson, S.A. in the Journal of Clinical Investigation, Vol. 39, Page 1157, 1960, described the first complete radioimmunoassay procedure. T. W. Smith et al. in the New England Journal of Medicine, Vol. 281, Page 1212, 1969, describes a radioimmunoassay for digoxin in which .sup.3 H-digoxin was used as the radioactive tracer, this tracer requiring a liquid scintillation counting fluid.